Compositions and methods of treating pulmonary hypertension

ABSTRACT

Provided are formulations comprising therapeutically effective amounts of ambrisentan or a pharmaceutically acceptable salt thereof and tadalafil or a pharmaceutically acceptable salt thereof and methods of treating and/or preventing pulmonary hypertension by administration of the formulations.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit and priority to U.S. ProvisionalApplication No. 61/393,529 filed Oct. 15, 2010, U.S. ProvisionalApplication No. 61/490,454 filed May 26, 2011, and U.S. ProvisionalApplication No. 61/497,475 filed Jun. 15, 2011. The entire disclosure ofthe applications identified in this paragraph is incorporated herein byreference.

FIELD

The present disclosure relates to methods of treating and/or preventingpulmonary hypertension by administration of therapeutically effectiveamounts of a selective type-A endothelin receptor antagonist and aphosphodiesterase type 5 inhibitor. This disclosure also relates topharmaceutical formulations that are suitable for such administration.

BACKGROUND

Pulmonary hypertension (PH) has been previously classified as primary(idiopathic) or secondary. Recently, the World Health Organization (WHO)has classified pulmonary hypertension into five groups:

Group 1: pulmonary arterial hypertension (PAH);

Group 2: PH with left heart disease;

Group 3: PH with lung disease and/or hypoxemia;

Group 4: PH due to chronic thrombotic and/or embolic disease; and

Group 5: miscellaneous conditions (e.g., sarcoidosis, histiocytosis X,lymphangiomatosis and compression of pulmonary vessels). See, forexample, Rubin (2004) Chest 126:7-10.

Pulmonary arterial hypertension (PAH) is a particular type of PH and isa serious, progressive and life-threatening disease of the pulmonaryvasculature, characterized by profound vasoconstriction and an abnormalproliferation of smooth muscle cells in the walls of the pulmonaryarteries. Severe constriction of the blood vessels in the lungs leads tovery high pulmonary arterial pressures. These high pressures make itdifficult for the heart to pump blood through the lungs to beoxygenated. Patients with PAH suffer from extreme shortness of breath asthe heart struggles to pump against these high pressures. Patients withPAH typically develop significant increases in pulmonary vascularresistance (PVR) and sustained elevations in pulmonary artery pressure(PAP), which ultimately lead to right ventricular failure and death.Patients diagnosed with PAH have a poor prognosis and equallycompromised quality of life, with a mean life expectancy of 2 to 5 yearsfrom the time of diagnosis if untreated.

Endothelin-1 (ET-1) is the primary member of a family of potentvasoconstrictor peptides, which are known to play an essential role inmammalian cardiovascular physiology. ET-1 is synthesized de novo andreleased from endothelial cells in response to a variety of factors,including angiotensin II, catecholamines, cytokines, hypoxia and shearstress. Two receptor subtypes, endothelin receptor type A (ET_(A)) andendothelin receptor type B (ET_(B)), mediate the effects of ET-1. Inhumans, the ET_(A) receptor is preferentially expressed in vascularsmooth muscle cells and is primarily responsible for thevasoconstrictive effects of ET-1. In contrast, ET_(B) receptors arefound mainly in the vascular endothelium, and their activation resultsin vasodilatation via production of nitric oxide and prostacyclin. TheET_(B) receptor is also involved in regulation of circulatingconcentrations of ET-1, through effects on endothelin converting enzyme(ECE-1) expression, and the synthesis and reuptake of ET-1 byendothelial cells.

Ambrisentan is a non-sulfonamide, propanoic acid-class endothelinreceptor antagonist (ERA) with high affinity (˜12 pM) for the ET_(A)receptor. Ambrisentan is approved for sale by the U.S. Food and DrugAdministration (FDA) for once-daily treatment of PAH and is marketedunder the trade name Letairis®. Other selective type-A receptorantagonists include sitaxentan, atrasentan, and BQ-123.

Additional drugs such as phosphodiesterase type 5 inhibitors (PDE5inhibitor) are also approved for use in treating PAH. PDE5 inhibitorsare drugs used to block the degradative action of phosphodiesterase type5 on cyclic GMP in the arterial wall smooth muscle within the lungs andin the smooth muscle cells lining the blood vessels supplying the corpuscavernosum of the penis.

Tadalafil is a PDE5 inhibitor, currently marketed under the nameAdcirca® for the treatment of pulmonary arterial hypertension. Theapproved dose for pulmonary arterial hypertension is 40 mg (two 20-mgtablets) once daily. Adverse effects of tadalafil include hypotension,vision loss, hearing loss and priapism. Thus, methods of increasing theanti-PH efficacy of selective type-A ERA and PDE5 inhibitors, as well asreducing the potential adverse effects, are highly desirable. Other PDE5inhibitors on the market or during development include avanafil,lodenafil, mirodenafil, sildenafil citrate, vardenafil and udenafil.

U.S. Patent Publication No. 2008/0139593 describes a method for treatingpulmonary hypertension, comprising administration of a therapeuticallyeffective amount of ambrisentan to a patient, wherein, at baseline, timefrom the first diagnosis of the condition in the subject is not greaterthan about two years. Also described is ambrisentan in combination withone or more suitable drugs selected from prostanoids, PDE5 inhibitorssuch as sildenafil, tadalafil, and vardenafil, ERAs, calcium channelblockers, arylalkylamines, dihydropyridine derivatives, piperazinederivatives and other suitable compounds for use in combination therapy.

It has now been discovered that the combination of a selective type-AERA and a PDE5 inhibitor has beneficial co-action resulting in potentrelaxation of pulmonary contractions. For example, the co-action ofambrisentan and tadalafil provides enhanced efficacy in reducingendothelin-induced contraction of rat pulmonary arteries and aortas.

SUMMARY OF THE DISCLOSURE

This disclosure describes the administration of a selective type-Aendothelin receptor antagonist (selective type-A ERA) in combinationwith a phosphodiesterase type 5 inhibitor (PDE5 inhibitor) which co-actsin relaxating pulmonary contractions and/or inhibiting hypoxia-inducedpulmonary arterial pressure (PAP). The ability to relax pulmonarycontraction or inhibit PAP is useful for treating and preventingpulmonary hypertension in patients, as well as a variety of otherconditions, which are described herein. This combination therapy leadsto enhanced therapeutic effects when the selective type-A ERA isadministered in a therapeutically effective dose and the PDE5 inhibitoris administered in a therapeutically effective dose. Either one or bothof the selective type-A ERA and the PDE5 inhibitor may be administeredin an amount less than their respective standard therapeutic doses dueto their co-action.

Certain ratios of the two agents even increase effectiveness of theco-action so that it is substantially greater than the sum ofeffectiveness of mono-administration of each agent (i.e., administrationof a single agent). In one aspect, the ratio of the amount of theselective type-A ERA and the amount of the PDE5 inhibitor, in order toget such enhanced effectiveness, can be from about 2:1 to about 1:3.Alternatively, the ratio of the amount of the selective type-A ERA andthe amount of the PDE5 inhibitor can range from 1:1, 1:1.5, 1:2, 1:2.5,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 to about 1:4, 1:5, 1:6, 1:7,1:8, 1:9, 1:10, 1:11, 1:12, 1:15 or 1:20. In another aspect, suchcombinations can achieve an effectiveness that is at least about 5%, oralternatively 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 80%, 90% or 100% greater than the sum of effectiveness ofmono-administration of each agent.

Non-limiting examples of selective type-A ERA include ambrisentan andsitaxentan and pharmaceutically acceptable salts thereof. In one aspect,the selective type-A ERA is ambrisentan. Examples of PDE5 inhibitorsinclude, without limitation, tadalafil, avanafil, lodenafil,mirodenafil, sildenafil citrate, vardenafil and udenafil andpharmaceutically acceptable salts thereof. In one aspect, the PDE5inhibitor is tadalafil.

Accordingly, in one aspect, this disclosure is directed to a method fortreatment and/or prevention of pulmonary hypertension in a patient inneed thereof. The method comprises administration of a therapeuticamount of ambrisentan or a salt thereof in combination with tadalafil ora salt thereof, wherein the ratio of the amount of ambrisentan or a saltthereof and the amount of tadalafil or a salt thereof is in a range fromabout 1:1 to about 1:10, or alternatively from about 1:2 to about 1:5,or alternatively about 1:3.

Another aspect of this disclosure provides a method for treating orpreventing pulmonary hypertension in a patient in need thereofcomprising administering to the patient therapeutic amounts ofambrisentan or a salt thereof in combination with tadalafil or a saltthereof, wherein the effectiveness of administration of the ambrisentanand the tadalafil is at least about 25%, or alternatively 40% or 50%,greater than the sum of effectiveness of mono-administrations of theambrisentan and the tadalafil.

Further provided, in one aspect, is a method for treating or preventingpulmonary hypertension in a patient in need thereof comprisingadministering to the patient, once daily, therapeutic amounts ofambrisentan or a salt thereof in combination with tadalafil or a saltthereof, wherein the ratio of the amount of ambrisentan or a saltthereof and the amount of tadalafil or a salt thereof is about 1:3.

In any of the embodiments described herein, the pulmonary hypertensioncomprises pulmonary arterial hypertension (PAH), including but notlimited to idiopathic PAH, familial PAH or PAH associated with anotherdisease or condition. In one aspect, the PAH at baseline is of WHO ClassI, II, III or IV.

In another aspect, provided is a method for inhibitingendothelin-induced vasoconstriction in a patient in need thereofcomprising administering to the patient therapeutic amounts ofambrisentan or a salt thereof in combination with tadalafil or a saltthereof, wherein (a) the ratio of the amount of ambrisentan or a saltthereof and the amount of tadalafil or a salt thereof is in a range fromabout 1:1 to about 1:10 and/or (b) the effectiveness of administrationof the ambrisentan and the tadalafil is at least about 25% greater thanthe sum of effectiveness of mono-administrations of the ambrisentan andthe tadalafil.

Also provided, in one aspect, is a method for treating or preventing adisease in a patient in need thereof comprising administering to thepatient therapeutic amounts of ambrisentan or a salt thereof incombination with tadalafil or a salt thereof, wherein the disease isselected from the group consisting of hypertension, pulmonaryhypertension, myocardial infarction, angina pectoris, acute kidneyfailure, renal insufficiency, cerebral vasospasms, cerebral ischemia,subarachnoid hemorrhages, asthma, atherosclerosis, intravascularcoagulation, restenosis after angioplasty, hypertension caused byischemia or intoxication, kidney failure caused by ischemia orintoxication, Raynaud's syndrome and asthmatic airway condition, andwherein (a) the ratio of the amount of ambrisentan or a salt thereof andthe amount of tadalafil or a salt thereof is in a range from about 1:1to about 1:10 and/or (b) the effectiveness of administration of theambrisentan and the tadalafil is at least about 25% greater than the sumof effectiveness of mono-administrations of the ambrisentan and thetadalafil.

Still further provided is a method for reducing undesirable side effectsof ambrisentan or a salt thereof comprising administering to the patienta therapeutic amount of tadalafil or a salt thereof, wherein (a) theratio of the amount of ambrisentan or a salt thereof and the amount oftadalafil or a salt thereof is in a range from about 1:1 to about 1:10and/or (b) the effectiveness of administration of the ambrisentan andthe tadalafil is at least about 25% greater than the sum ofeffectiveness of mono-administrations of the ambrisentan and thetadalafil.

A method for reducing a therapeutically effective dose of ambrisentan ora salt thereof is also provided, the method comprising administering tothe patient a therapeutic amount of tadalafil or a salt thereof, wherein(a) the ratio of the amount of ambrisentan or a salt thereof and theamount of tadalafil or a salt thereof is in a range from about 1:1 toabout 1:10 and/or (b) the effectiveness of administration of theambrisentan and the tadalafil is at least about 25% greater than the sumof effectiveness of mono-administrations of the ambrisentan and thetadalafil.

Yet in one aspect, this disclosure provides a method for reducingundesirable side effects of tadalafil or a salt thereof comprisingadministering to the patient a therapeutic amount of ambrisentan or asalt thereof, wherein (a) the ratio of the amount of ambrisentan or asalt thereof and the amount of tadalafil or a salt thereof is in a rangefrom about 1:1 to about 1:10 and/or (b) the effectiveness ofadministration of the ambrisentan and the tadalafil is at least about25% greater than the sum of effectiveness of mono-administrations of theambrisentan and the tadalafil.

In another aspect, provided is a method for reducing a therapeuticallyeffective dose of tadalafil or a salt thereof comprising administeringto the patient a therapeutic amount of ambrisentan or a salt thereof,wherein (a) the ratio of the amount of ambrisentan or a salt thereof andthe amount of tadalafil or a salt thereof is in a range from about 1:1to about 1:10 and/or (b) the effectiveness of administration of theambrisentan and the tadalafil is at least about 25% greater than the sumof effectiveness of mono-administrations of the ambrisentan and thetadalafil.

Pharmaceutical formulations are also provided. One aspect provides apharmaceutical formulation comprising therapeutic amounts of tadalafilor a salt thereof and ambrisentan or a salt thereof, and apharmaceutically acceptable carrier, wherein (a) the ratio of the amountof ambrisentan or a salt thereof and the amount of tadalafil or a saltthereof is in a range from about 1:1 to about 1:10 and/or (b) theeffectiveness of combination of the ambrisentan and the tadalafil is atleast about 25% greater than the sum of effectiveness ofmono-administrations of the ambrisentan and the tadalafil.

In one aspect, this disclosure provides a method for treating pulmonaryhypertension in a patient comprising administering a therapeuticallyeffective amount of a combination therapy comprising of a selectivetype-A endothelin receptor antagonist, in combination with a PDE5inhibitor and metabolites thereof, co-acting to provide therapeuticbenefit to patients with pulmonary hypertension in absence of asubstantial deleterious side effect in a therapeutically effective ratioin a range from about 1:1 to about 1:10.

In another aspect, this invention discloses a method for treatingpulmonary hypertension in a patient comprising administering atherapeutically effective amount of a combination therapy comprising ofa selective type-A endothelin receptor antagonist, a PDE5 inhibitor, anda third active agent effective for the treatment of pulmonaryhypertension or a related condition, and metabolites thereof, co-actingto provide therapeutic benefit to patients with pulmonary hypertensionin absence of a substantial deleterious side effect in a therapeuticallyeffective molar ratio in a range from about 1:1 to about 1:10.

In one aspect, this disclosure provides a pharmaceutical formulation ofa combination therapy comprising therapeutic amounts of a selectivetype-A endothelin receptor antagonist and a PDE5 inhibitor co-acting toprovide therapeutic benefit to patients with pulmonary hypertension inabsence of a substantial deleterious side effect in a therapeuticallyeffective ratio in a range from about 1:1 to about 1:10.

In one aspect, this disclosure provides a pharmaceutical formulationcomprising therapeutic amounts of tadalafil or a salt thereof andambrisentan or a salt thereof, and a pharmaceutically acceptablecarrier, wherein the ratio of the amount of ambrisentan or a saltthereof and the amount of tadalafil or a salt thereof is about 1:3.

In another aspect, this disclosure provides a pharmaceutical formulationof a combination therapy comprising a selective type-A endothelinreceptor antagonist, a PDE5 inhibitor, and a third active agenteffective for the treatment of pulmonary hypertension or a relatedcondition, and metabolites thereof, co-acting to provide therapeuticbenefit to patients with pulmonary hypertension in absence of asubstantial deleterious side effect in a therapeutically effective molarratio in a range from about 1:1 to about 1:10.

BRIEF DESCRIPTION OF THE DRAWINGS

As used throughout the Figures, the term “AMB” refers to ambrisentan,“TAD” refers to tadalafil, “BOS” refers to bosentan, and “MAC” refers tomacitentan.

FIG. 1 shows that ambrisentan (10 nM) and tadalafil (30 nM), incombination, relaxed endothelin-induced contraction of rat pulmonaryarteries and aortas significantly more effectively thanmono-administration of either drug. Data are expressed as mean±SEM, n=3.*p<0.01 vs. mono-therapy of 10 nM Ambrisentan or 30 nM tadalafil.

FIG. 2 shows that the combination of ambrisentan and tadalafil exhibitedmore than additive effects than mono-therapy of either drug, and thus,beneficial co-action exists between ambrisentan and tadalafil. Suchco-action, however, was not observed between non-selective ERA, such asbosentan and macitentan, when administered with tadalafil. *p<0.01 vs.Ambrisentan (AMB) or Tadalafil (TAD). #p<0.05 vs. mono-administrationsof Bosentan (BOS), Macitentan (MAC) and TAD or combination of ABM withTAD. - - - : represents the predicted additive effect of AMB with TAD.

FIG. 3 shows the effects of TAD with AMB or BOS to attenuateET-1-induced contraction of rat aortic rings. Data are expressed asmean±SEM. *p<0.01 vs. 10 nM AMB or 30 nM TAD. #p<0.05 vs. 100 nM BOS or30n M TAD. - - - : represents the additive effect of AMB with TAD. Thedata show that the effect of the combination of AMB and TAD is greaterthan the additive effects of each drug (indicated by the dotted line),whereas the combinatory effect of BOS and TAD is not.

FIG. 4 are contraction graphs showing the effects of TAD in combinationwith AMB or BOS on ET-1-induced contraction of rat aortic rings (top: notreatment; middle: AMB+TAD; bottom: BOS+TAD). It is shown that thecombination of AMB and TAD (middle) relaxed contraction moresignificantly than the combination of BOS and TAD (bottom).

FIG. 5 shows that endothelium involved in co-action or additive effectof ET Receptor antagonists with TAD.

FIG. 6 shows that in the presence of BQ-788, a selective type-Bendothelin receptor antagonist, the combination effect of AMB with TADwas significantly reduced. This indicates that ET type-B receptor isinvolved in co-action effect of AMB with TAD.

FIG. 7 illustrates the ET-1 and PDE5 signaling in endothelial cells andsmooth muscle cells.

FIG. 8 illustrates the mechanism underlying the co-action between aselective type-A ERA and a PDE5 inhibitor that target vasorelaxationfrom two different pathways.

FIG. 9 illustrates the lack of beneficial co-action between anon-selective ERA or a selective type-B ERA and a PDE5 inhibitor thattarget the same pathway.

FIG. 10 demonstrates the co-action between TAD with AMB in an in vivopulmonary arterial hypertension (PAH) animal model.

DETAILED DESCRIPTION OF THE DISCLOSURE 1. Definitions and GeneralParameters

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

It is to be noted that as used herein and in the claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “apharmaceutically acceptable carrier” in a composition includes two ormore pharmaceutically acceptable carriers, and so forth.

“Comprising” is intended to mean that the compositions and methodsinclude the recited elements, but do not exclude others. “Consistingessentially of” when used to define compositions and methods, shall meanexcluding other elements of any essential significance to thecombination for the intended use. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this disclosure. Embodimentsdefined by each of these transition terms are within the scope of thisdisclosure.

An “endothelin receptor antagonist (ERA)” is an agent that blocksendothelin receptors. There are at least two major known endothelinreceptors, ET_(A) and ET_(B), both of which are G protein-coupledreceptors whose activation result in elevation of intracellular-freecalcium. Three main kinds of ERAs exist: selective type-A receptorantagonists, e.g., sitaxentan, ambrisentan, atrasentan, BQ-123, whichaffect endothelin A receptors; dual antagonists, e.g., bosentan,macitentan, tezosentan, which affect both endothelin A and B receptors;and selective type-B receptor antagonists, e.g., BQ-788, which affectendothelin B receptors.

A “selective type-A endothelin receptor antagonist” or “selective type-AERA” selectively targets the type-A endothelin receptor.

“Ambrisentan” or “AMB” is described in U.S. Pat. Nos: 5,703,017;5,932,730 and 7,109,205. It refers to the chemical compound,(2S)-2-[(4,6-dimethylpyrimidin-2-yl)oxy]-3-methoxy-3,3-diphenylpropanoicacid and has the following chemical formula:

Ambrisentan is approved for sale by the U.S. Food and DrugAdministration (FDA) for once-daily treatment of PAH and is marketedunder the trade name Letairis®. In Europe, Ambrisentan is approved underthe trade name Volibris®.

“Ambrisentan” as used herein is intended to include the metabolites ofambrisentan described in U.S. Patent Publication No. 2010/0204163. Theambrisentan metabolites include the compounds having the followingchemical formula:

wherein R¹ is —OH or —OCH₃; R² is —H, lower alkyl or glycosidyl; and R³and R⁴ are independently —CH₃, —C(O)H or —CH₂OR⁶, wherein R⁶ is —H or ahydrocarbyl group having 1 to 20 carbon atoms.

“Sitaxentan” refers to the chemical compoundN-(4-chloro-3-methyl-1,2-oxazol-5-yl)-2-[2-(6-methyl-2H-1,3-benzodioxol-5-yl)acetyl]thiophene-3-sulfonamide,and its pharmaceutically acceptable salts. Sitaxentan is described inBarst R J et al., (2004) American Journal of Respiratory Critical CareMedicine 169 (4): 441-7. Sitaxentan has the following chemical formula:

Sitaxentan was marketed as Thelin® for the treatment of PAH in 2008 butwas later removed from the market in 2010.

A “phosphodiesterase type 5 inhibitor” or “PDE5 inhibitor” refers to anagent that blocks the degradative action of phosphodiesterase type 5 oncyclic GMP in the arterial wall smooth muscle within the lungs and inthe smooth muscle cells lining the blood vessels supplying the corpuscavernosum of the penis. PDE5 inhibitors are used for the treatment ofpulmonary hypertension and in the treatment of erectile dysfunction.Examples of PDE5 inhibitors include, without limitation, tadalafil,avanafil, lodenafil, mirodenafil, sildenafil citrate, vardenafil andudenafil and pharmaceutically acceptable salts thereof. In one aspect,the PDE5 inhibitor is tadalafil.

“Tadalafil” or “TAD” is described in U.S. Pat. Nos. 5,859,006 and6,821,975. It refers to the chemical compound,(6R-trans)-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methyl-pyrazino[1′, 2′:1,6]pyrido[3,4-b]indole-1,4-dione and has the following chemicalformula:

Tadalafil is currently marketed in pill form for treating erectiledysfunction (ED) under the trade name Cialis® and under the trade nameAdcirca® for the treatment of PAH.

“Avanafil” refers to the chemical compound4-[(3-Chloro-4-methoxybenzyl)amino]-2-[2-(hydroxymethyl)-1-pyrrolidinyl]-N-(2-pyrimidinylmethyl)-5-pyrimidinecarboxamide,and its pharmaceutically acceptable salts. Avanafil is described inLimin M. et al., (2010) Expert Opin Investig Drugs, 19(11):1427-37.Avanafil has the following chemical formula:

Avanafil is being developed for erectile dysfunction. Avanafil currentlyhas no trademarked term associated with it but it is being developed byVivus Inc.

“Lodenafil” refers to the chemical compound,bis-(2-{4-[4-ethoxy-3-(1-methyl-7-oxo-3propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-benzenesulfonyl]piperazin-1-yl}-ethyl)carbonateand has the following chemical formula:

More information about lodenafil is available at Toque H A et al.,(2008) European Journal of Pharmacology, 591(1-3):189-95. Lodenafil ismanufactured by Cristália Produtos Químicos e Farmacéuticos in Braziland sold there under the brand-name Helleva®. It has undergone Phase IIIclinical trials, but is not yet approved for use in the United States bythe U.S. FDA.

“Mirodenafil” refers to the chemical compound,5-Ethyl-3,5-dihydro-2-[5-([4-(2-hydroxyethyl)-1-piperazinyl]sulfonyl)-2-propoxyphenyl]-7-propyl-4H-pyrrolo[3,2-d]pyrimidin-4-oneand has the following chemical formula:

More information about mirodenafil can be found at Paick J S et al.,(2008) The Journal of Sexual Medicine, 5 (11): 2672-80. Mirodenafil isnot currently approved for use in the United States but clinical trialsare being conducted.

“Sildenafil citrate,” marketed under the name Viagra®, is described inU.S. Pat. No. 5,250,534. It refers to1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)phenylsulfonyl]-4-methylpiperazineand has the following chemical formula:

Sildenafil citrate, sold as Viagra®, Revatio® and under various othertrade names, is indicated to treat erectile dysfunction and PAH.

“Vardenafil” refers to the chemical compound,4-[2-Ethoxy-5-(4-ethylpiperazin-1-yl)sulfonyl-phenyl]-9-methyl-7-propyl-3,5,6,8-tetrazabicyclo[4.3.0]nona-3,7,9-trien-2-oneand has the following chemical formula:

Vardenafil is described in U.S. Pat. Nos. 6,362,178 and 7,696,206.Vardenafil is marketed under the trade name Levitra® for treatingerectile dysfunction.

“Udenafil” refers to the chemical compound,3-(1-methyl-7-oxo-3-propyl-4,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamideand has the following chemical formula:

More information about udenafil can be found at Kouvelas D. et al.,(2009) Curr Pharm Des, 15(30):3464-75. Udenafil is marketed under thetrade name Zydena® but not approved for use in the United States.

Each of the compounds described above, as used throughout, is intendedto include a free acid, free base, or a pharmaceutically acceptable saltthereof.

As used herein, the term “salt” refers to a pharmaceutically acceptablesalt of a compound that is derived from a variety of physiologicallyacceptable organic and inorganic counter ions. Such counter ions arewell known in the art and include, by way of example only, sodium,potassium, calcium, magnesium, aluminum, lithium and ammonium, forexample tetraalkylammonium, and the like when the molecule contains anacidic functionality; and when the molecule contains a basicfunctionality, salts of organic or inorganic acids, such ashydrochloride, sulfate, phosphate, diphosphate, nitrate hydrobromide,tartrate, mesylate, acetate, malate, maleate, besylate, fumarate,tartrate, succinate, citrate, lactate, pamoate, salicylate, stearate,methanesulfonate, p-toluenesulfonate, and oxalate, and the like.Suitable pharmaceutically acceptable salts also include those listed inRemington's Pharmaceutical Sciences, 17th Edition, pg. 1418 (1985) andP. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of PharmaceuticalSalts Properties, Selection, and Use; 2002. Examples of acid additionsalts include those formed from acids such as hydroiodic, phosphoric,metaphosphoric, nitric and sulfuric acids, and with organic acids, suchas alginic, ascorbic, anthranilic, benzoic, camphorsulfuric, citric,embonic (pamoic), ethanesulfonic, formic, fumaric, furoic, galacturonic,gentisic, gluconic, glucuronic, glutamic, glycolic, isonicotinic,isothionic, lactic, malic, mandelic, methanesulfonic, mucic,pantothenic, phenylacetic, propionic, saccharic, salicylic, stearic,succinic, sulfinilic, trifluoroacetic and arylsulfonic for examplebenzenesulfonic and p-toluenesulfonic acids. Examples of base additionsalts formed with alkali metals and alkaline earth metals and organicbases include chloroprocaine, choline, N,N-dibenzylethylenediamine,diethanolamine, ethylenediamine, lysine, meglumaine (N-methylglucamine),and procaine, as well as internally formed salts. Salts having anon-physiologically acceptable anion or cation are within the scope ofthe disclosure as useful intermediates for the preparation ofphysiologically acceptable salts and/or for use in non-therapeutic, forexample, in vitro, situations.

The disclosure specifically contemplates using salts of both ambrisentanand tadalafil and further contemplates mixtures of salts of tadalafiland/or ambrisentan.

In certain embodiments, the ambrisentan and/or tadalafil as used hereinhas not been sufficiently ionized and may be in the form a co-crystal.In one embodiment, the present disclosure provides a co-crystalcomposition comprising a co-crystal of ambrisentan and/or tadalafil,wherein said co-crystal comprises ambrisentan and/or tadalafil and aco-crystal former. The term “co-crystal” refers a crystalline materialwhich comprises ambrisentan and/or tadalafil and one or more co-crystalformers, such as a pharmaceutically acceptable salt. In certainembodiments, the co-crystal can have an improved property as compared tothe free form (i.e., the free molecule, zwitter ion, hydrate, solvate,etc.) or a salt (which includes salt hydrates and solvates). In furtherembodiments, the improved property is selected from the group consistingof: increased solubility, increased dissolution, increasedbioavailability, increased dose response, decreased hygroscopicity, acrystalline form of a normally amorphous compound, a crystalline form ofa difficult to salt or unsaltable compound, decreased form diversity,more desired morphology, and the like. Methods for making andcharacterizing co-crystals are well known to those of skill in the art.

The phrase “combination therapy”, in defining use of a selective type-Aendothelin receptor antagonist and a PDE5 inhibitor, is intended toembrace administration of each agent in a sequential manner in a regimenthat will provide beneficial effects of the drug combination, and isintended as well to embrace co-administration of these agents in asubstantially simultaneous manner, such as by oral ingestion of a singlecapsule having a fixed ratio of these active agents or ingestion ofmultiple, separate capsules for each agent. “Combination therapy” willalso include simultaneous or sequential administration by intravenous,intramuscular or other parenteral routes into the body, including directabsorption through mucous membrane tissues, as found in the sinuspassages. Sequential administration also includes drug combination wherethe individual elements may be administered at different times and/or bydifferent routes but which act in combination to provide a beneficialeffect, such as enhanced effectiveness.

In a particular embodiment, a combination therapy consists essentiallyof two active agents, namely, a selective type-A endothelin receptorantagonist and a PDE5 inhibitor.

In another embodiment, a combination therapy is a three-way combinationcomprising a selective type-A endothelin receptor antagonist, a PDE5inhibitor and a third active agent effective for the treatment of thepulmonary hypertension condition or a condition related thereto.Illustratively and without limitation, the combination can include athird active agent selected from the group consisting of prostanoids,phosphodiesterase inhibitors other than tadalafil, endothelin receptorantagonists other than ambrisentan, calcium channel blockers, diuretics,anticoagulants, oxygen and combinations thereof.

The phrase “therapeutically-effective” is intended to qualify the amountof each agent for use in the combination therapy which will achieve thegoal of improvement in pulmonary functions, while avoiding or reducingan adverse side effect typically associated with each agent. Thetherapeutically effective amount will vary depending upon the specificactivity of the therapeutic agent being used, the severity of thepatient's disease state, and the age, physical condition, existence ofother disease states, and nutritional status of the patient.Additionally, other medication the patient may be receiving will effectthe determination of the therapeutically effective amount of thetherapeutic agent to administer.

“Co-action” means that the therapeutic effect of a PDE5 inhibitor suchas tadalafil when administered in combination with a selective type-Aendothelin receptor antagonist such as ambrisentan (or vice-versa) isgreater than the sum of the therapeutic effects of the agents whenadministered separately. The term “therapeutic amount” used hereinincludes a less than standard therapeutic amount of one or both drugs,meaning that the amount required for the desired effect is lower thanwhen the drug is used separately. A therapeutic amount also includeswhen one drug is given at a standard therapeutic dose and another drugis administered in a less than standard therapeutic dose. For example,ambrisentan could be given in a therapeutic dose and tadalafil could begiven in a less than standard therapeutic dose to provide an enhancedresult. In some embodiments, both drugs can be administered in astandard therapeutic dose for much greater efficacies.

The term “treatment” or “treating” means any treatment of a disease orcondition in a subject, such as a mammal, including: 1) preventing orprotecting against the disease or condition, that is, causing theclinical symptoms not to develop; 2) inhibiting the disease orcondition, that is, arresting or suppressing the development of clinicalsymptoms; and/or 3) relieving the disease or condition that is, causingthe regression of clinical symptoms.

As used herein, the term “preventing” refers to the prophylactictreatment of a patient in need thereof. The prophylactic treatment canbe accomplished by providing an appropriate dose of a therapeutic agentto a subject at risk of suffering from an ailment, thereby substantiallyaverting onset of the ailment.

It will be understood by those skilled in the art that in humanmedicine, it is not always possible to distinguish between “preventing”and “suppressing” since the ultimate inductive event or events may beunknown, latent, or the patient is not ascertained until well after theoccurrence of the event or events. Therefore, as used herein the term“prophylaxis” is intended as an element of “treatment” to encompass both“preventing” and “suppressing” as defined herein. The term “protection,”as used herein, is meant to include “prophylaxis.”

The term “susceptible” refers to a patient who has had at least oneoccurrence of the indicated condition.

The term “patient” typically refers to a “mammal” which includes,without limitation, human, monkeys, rabbits, mice, domestic animals,such as dogs and cats, farm animals, such as cows, horses, or pigs, andlaboratory animals.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

“Intravenous administration” is the administration of substancesdirectly into a vein, or “intravenously”. Compared with other routes ofadministration, the intravenous (IV) route is the fastest way to deliverfluids and medications throughout the body. An infusion pump can allowprecise control over the flow rate and total amount delivered, but incases where a change in the flow rate would not have seriousconsequences, or if pumps are not available, the drip is often left toflow simply by placing the bag above the level of the patient and usingthe clamp to regulate the rate. Alternatively, a rapid infuser can beused if the patient requires a high flow rate and the IV access deviceis of a large enough diameter to accommodate it. This is either aninflatable cuff placed around the fluid bag to force the fluid into thepatient or a similar electrical device that may also heat the fluidbeing infused. When a patient requires medications only at certaintimes, intermittent infusion is used, which does not require additionalfluid. It can use the same techniques as an intravenous drip (pump orgravity drip), but after the complete dose of medication has been given,the tubing is disconnected from the IV access device. Some medicationsare also given by IV push or bolus, meaning that a syringe is connectedto the IV access device and the medication is injected directly (slowly,if it might irritate the vein or cause a too-rapid effect). Once amedicine has been injected into the fluid stream of the IV tubing theremust be some means of ensuring that it gets from the tubing to thepatient. Usually this is accomplished by allowing the fluid stream toflow normally and thereby carry the medicine into the bloodstream;however, a second fluid injection is sometimes used, a “flush”,following the injection to push the medicine into the bloodstream morequickly.

“Oral administration” is a route of administration where a substance istaken through the mouth, and includes buccal, sublabial and sublingualadministration, as well as enteral administration and that through therespiratory tract, unless made through, e.g., tubing so the medicationis not in direct contact with any of the oral mucosa. Typical form forthe oral administration of therapeutic agents includes the use oftablets or capsules.

2. Methods

Generally, the present disclosure relates to methods of treating orpreventing pulmonary hypertension. The method comprises administrationof therapeutic amounts of a selective type-A endothelin receptorantagonist (selective type-A ERA) and a phosphodiesterase type 5inhibitor (PDE5 inhibitor). In a particular aspect, the method comprisesadministration of a therapeutic amount of tadalafil or a salt thereofand a therapeutic amount of ambrisentan or a salt thereof. In oneembodiment, either one or both of ambrisentan or tadalafil areadministered in an effective amount. The two agents may be administeredseparately or together in separate or a combined dosage unit. Ifadministered separately, the ambrisentan may be administered before orafter administration of the tadalafil.

As further discussed in the Examples, presented herewith is evidence ofco-action of the combination of ambrisentan (AMB) and tadalafil (TAD) torelax endothelin-induced contractions and to inhibit hypoxia-inducedpulmonary arterial pressure (PAP) in a pulmonary arterial hypertension(PAH) animal model. Such enhanced efficacy of the co-action is apparentas the combined effect is greater than the additive effects ofmono-administration of each drug. In one aspect, such enhanced efficacyamounts to at least about 5% enhanced effectiveness over the additiveeffectiveness of mono-administration of each drug. Alternatively, suchenhancement is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 80%, 90% or 100%. In other words, thecombinations can achieve an effectiveness that is at least about 5%, oralternatively 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 80%, 90% or 100% greater than the sum of effectiveness ofmono-administrations of either agent.

As demonstrated in the Examples, such an enhanced effect does not existbetween TAD and a non-selective ERA such as bosentan (BOS) or macitentan(MAC). These results, therefore, suggest that type-B endothelin receptorin endothelium contributes to the enhanced effect of ambrisentan andtadalafil on vasorelaxation. As illustrated in FIGS. 8 and 9, theenhanced effect is due to ambrisentan and tadalafil co-acting in theendothelin receptor type-A and PDE5 pathways, respectively. Accordingly,similar enhanced effects are found between any selective type-A ERA andany PDE5 inhibitor. Additionally, a combination of a selective type-AERA and a PDE5 inhibitor also results in enhanced effects in treatingother diseases and conditions associated with the activity of endothelinreceptor type-A.

As suggested by the above mechanism, the enhanced effect of theco-action of a selective type-A ERA and a PDE5 inhibitor depends on theamounts of each individual agent and/or ratios of such amounts. In oneaspect, the ratio of the amount of the selective type-A ERA and theamount of the PDE5 inhibitor, in order to achieve such enhanced effects,can be from about 2:1, or alternatively 1:1, 1:1.5, 1:2, 1:2.5, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 to about 1:3, or alternativelyabout 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:15 or 1:20.

In one aspect, the ratio of amounts is a ratio of molar amounts of eachagent. In another aspect, the ratio of amounts is a weight ratio of eachagent.

In some embodiments, the ratio of the amount of the selective type-A ERAand the amount of the PDE5 inhibitor, in order to achieve enhancedeffects, is around 1:3, which, for instance, can be from about 1:1.5 toabout 1:5, or alternatively from about 1:2 to about 1:4. In one aspect,the selective type-A ERA is ambrisentan or a salt thereof In one aspect,the PDE5 inhibitor is tadalafil or a salt thereof. In another aspect,the ratio is a weight ratio of each agent. In one aspect, the amount ofambrisentan or a salt thereof is from about 5 mg to about 10 mg dailyfor a human subject. In another aspect, the amount of tadalafil or asalt thereof is from about 15 mg to about 30 mg daily for a humansubject.

In some embodiments, the ratio of the amount of the selective type-A ERAand the amount of the PDE5 inhibitor, in order to achieve enhancedeffects, is around 1:1, which, for instance, can be from about 2:1 toabout 1:2, or alternatively from about 1:1 to about 1:2. In one aspect,the selective type-A ERA is ambrisentan or a salt thereof. In oneaspect, the PDE5 inhibitor is tadalafil or a salt thereof In anotheraspect, the ratio is a weight ratio of each agent. In one aspect, theamount of ambrisentan or a salt thereof is from about 5 mg to about 10mg daily for a human subject. In another aspect, the amount of tadalafilor a salt thereof is from about 5 mg to about 10 mg daily for a humansubject.

In some embodiments, the ratio of the amount of the selective type-A ERAand the amount of the PDE5 inhibitor, in order to achieve enhancedeffects, is around 1:10, which, for instance, can be from about 1:5 toabout 1:15, or alternatively from about 1:8 to about 1:12. In oneaspect, the selective type-A ERA is ambrisentan or a salt thereof In oneaspect, the PDE5 inhibitor is tadalafil or a salt thereof. In anotheraspect, the ratio is a weight ratio of each agent. In one aspect, theamount of ambrisentan or a salt thereof is from about 2 mg to about 5 mgdaily for a human subject. In another aspect, the amount of tadalafil ora salt thereof is from about 20 mg to about 40 mg daily for a humansubject.

In some embodiments, the ratio of the amount of the selective type-A ERAand the amount of the PDE5 inhibitor, in order to achieve enhancedeffects, is around 1:4, which, for instance, can be from about 1:2 toabout 1:7, or alternatively from about 1:3 to about 1:5. In one aspect,the selective type-A ERA is ambrisentan or a salt thereof. In oneaspect, the PDE5 inhibitor is tadalafil or a salt thereof. In anotheraspect, the ratio is a weight ratio of each agent. In one aspect, theamount of ambrisentan or a salt thereof is from about 5 mg to about 10mg daily for a human subject. In another aspect, the amount of tadalafilor a salt thereof is from about 30 mg to about 40 mg daily for a humansubject.

In some embodiments, the ratio of the amount of the selective type-A ERAand the amount of the PDE5 inhibitor, in order to achieve enhancedeffects, is around 1:8, which, for instance, can be from about 1:5 toabout 1:10, or alternatively from about 1:7 to about 1:9. In one aspect,the selective type-A ERA is ambrisentan or a salt thereof In one aspect,the PDE5 inhibitor is tadalafil or a salt thereof. In another aspect,the ratio is a weight ratio of each agent. In one aspect, the amount ofambrisentan or a salt thereof is from about 2 mg to about 5 mg daily fora human subject. In another aspect, the amount of tadalafil or a saltthereof is from about 30 mg to about 40 mg daily for a human subject.

Non-limiting examples of selective type-A ERA include ambrisentan andsitaxentan and salts thereof. In one aspect, the selective type-A ERA isambrisentan. Examples of PDE5 inhibitors include, without limitation,tadalafil, avanafil, lodenafil, mirodenafil, sildenafil citrate,vardenafil and udenafil and salts thereof. In one aspect, the PDE5inhibitor is tadalafil.

Non-limiting examples of disease or condition associated with theactivity of endothelin receptor type-A include hypertension, pulmonaryhypertension, myocardial infarction, angina pectoris, acute kidneyfailure, renal insufficiency, cerebral vasospasms, cerebral ischemia,subarachnoid hemorrhages, asthma, atherosclerosis, intravascularcoagulation, restenosis after angioplasty, hypertension caused byischemia or intoxication, kidney failure caused by ischemia orintoxication, Raynaud's syndrome and asthmatic airway condition.

2.1 Pulmonary Hypertension (PH)

The pulmonary hypertension condition treated by the method of thedisclosure, can comprise any one or more of the conditions recognizedaccording to the World Health Organization (WHO) or Venice (2003)classification (see, for example, Rubin (2004) Chest 126:7-10) or themost recent Dana Point classification (Simonneau (2009) JACC 54;54:S43-S54):

Group 1: Pulmonary Arterial Hypertension (PAH)

-   -   1.1 idiopathic PAH    -   1.2 familial PAH    -   1.3 PAH associated with:    -   1.3.1 collagen vascular disease    -   1.3.2 congenital systemic-to-pulmonary shunts (including        Eisenmenger's syndrome)    -   1.3.3 portal hypertension    -   1.3.4 HIV infection    -   1.3.5 drugs and toxins    -   1.3.6 other (thyroid disorders, glycogen storage disease,        Gaucher disease, hereditary hemorrhagic telangiectasia,        hemoglobinopathies, myeloproliferative disorders, splenectomy)    -   1.4 PAH associated with significant venous or capillary        involvement    -   1.4.1 pulmonary veno-occlusive disease (PVOD)    -   1.4.2 pulmonary capillary hemangiomatosis (PCH)    -   1.5 persistent pulmonary hypertension of the newborn

Group 2: Pulmonary hypertension with left heart disease

-   -   2.1 left-sided atrial or ventricular heart disease    -   2.2 left-sided valvular heart disease

Group 3: Pulmonary hypertension associated with lung diseases and/orhypoxemia

-   -   3.1 chronic obstructive pulmonary disease (COPD)    -   3.2 interstitial lung disease    -   3.3 sleep-disordered breathing    -   3.4 alveolar hypoventilation disorders    -   3.5 chronic exposure to high altitude    -   3.6 developmental abnormalities

Group 4: Pulmonary hypertension due to chronic thrombotic and/or embolicdisease

-   -   4.1 thromboembolic obstruction of proximal pulmonary arteries    -   4.2 thromboembolic obstruction of distal pulmonary arteries    -   4.3 non-thrombotic pulmonary embolism (tumor, parasites, foreign        material)

Group 5: Miscellaneous (sarcoidosis, histiocytosis X, lymphangiomatosis,compression of pulmonary vessels (adenopathy, tumor, fibrosingmediastinitis))

In one aspect, the pulmonary hypertension condition comprises PAH (WHOGroup 1), for example idiopathic PAH, familial PAH or PAH associatedwith another disease or condition.

Pulmonary hypertension at baseline can be mild, moderate or severe, asmeasured for example by WHO functional class, which is a measure ofdisease severity in patients with pulmonary hypertension. The WHOfunctional classification is an adaptation of the New York HeartAssociation (NYHA) system and is routinely used to qualitatively assessactivity tolerance, for example in monitoring disease progression andresponse to treatment (Rubin (2004) Chest 126:7-10). Four functionalclasses are recognized in the WHO system:

Class I: pulmonary hypertension without resulting limitation of physicalactivity; ordinary physical activity does not cause undue dyspnea orfatigue, chest pain or near syncope;

Class II: pulmonary hypertension resulting in slight limitation ofphysical activity; patient comfortable at rest; ordinary physicalactivity causes undue dyspnea or fatigue, chest pain or near syncope;

Class III: pulmonary hypertension resulting in marked limitation ofphysical activity; patient comfortable at rest; less than ordinaryactivity causes undue dyspnea or fatigue, chest pain or near syncope;

Class IV: pulmonary hypertension resulting in inability to carry out anyphysical activity without symptoms; patient manifests signs ofright-heart failure; dyspnea and/or fatigue may be present even at rest;discomfort is increased by any physical activity.

In one aspect, the subject at baseline exhibits pulmonary hypertension(e.g., PAH) of at least WHO Class I, for example WHO Class I, II orClass III.

In another aspect, the subject at baseline exhibits mean PAP at rest ofat least about 30 mmHg, for example at least about 35, at least about40, at least about 45 or at least about 50 mmHg.

The methods of the present disclosure, when applied to a subject, canachieve one or more of the following objectives:

(a) adjustment of one or more hemodynamic parameters towards a morenormal level, for example lowering mean PAP or PVR, raising cardiacoutput or index, or lowerin PCWP or LVEDP, versus baseline;

(b) improvement of pulmonary function versus baseline, for exampleincreasing exercise capacity or activity, illustratively as measured ina test of 6-minute walking distance (6MWD) or measure of activity, orlowering Borg dyspnea index (BDI);

(c) improvement of one or more quality of life parameters versusbaseline, for example an increase in score on at least one of the SF-36®health survey functional scales;

(d) general improvement versus baseline in the severity of thecondition, for example by movement to a lower WHO functional class;

(e) improvement of clinical outcome following a period of treatment,versus expectation in absence of treatment (e.g., in a clinical trialsetting, as measured by comparison with placebo), including improvedprognosis, extending time to or lowering probability of clinicalworsening, extending quality of life (e.g., delaying progression to ahigher WHO functional class or slowing decline in one or more quality oflife parameters such as SF-36® health survey parameters), and/orincreasing longevity; and/or

(f) adjustment towards a more normal level of one or more molecularmarkers that can be predictive of clinical outcome (e.g., plasmaconcentrations of endothelin-1 (ET-1), cardiac troponin T (cTnT) orB-type natriuretic peptide (BNP)).

What constitutes a therapeutically effective amount for treating PH, orin particular, PAH, can vary depending on the particular pulmonaryhypertension condition to be treated, the severity of the condition,body weight and other parameters of the individual subject, and can bereadily established without undue experimentation by the physician orclinician based on the disclosure herein.

Various clinical parameters and standards to measure the effectivenessof a PH therapy are described below and are known in the art as well.Accordingly, the effectiveness of a PH therapy, such as that of anycombination formulation of the present disclosure, can be measured bythese parameters or standards. Additionally, the relative effectivenessof a therapy, such as that of a combination of two agents, as comparedto the effectiveness of mono-administrations of each agent, can bedetermined with these clinical parameters or standards, as well as in anon-clinical setting. Examples of such non-clinical settings include,without limitation, an in vitro assay or animal study. Non-limitingexamples of in vitro assays are provided in Examples.

A. Improvement on Clinical Parameters

In one aspect, the subject being treated experiences, during orfollowing the treatment period, at least one of

(a) adjustment of one or more hemodynamic parameters indicative of thepulmonary hypertension condition towards a more normal level versusbaseline;

(b) increase in exercise capacity versus baseline;

(c) lowering of BDI versus baseline;

(d) improvement of one or more quality of life parameters versusbaseline; and/or

(e) movement to a lower WHO functional class.

Any suitable measure of exercise capacity can be used; a particularlysuitable measure is obtained in a 6-minute walk test (6MWT), whichmeasures how far the subject can walk in 6 minutes, i.e., the 6-minutewalk distance (6MWD).

The Borg dyspnea index (BDI) is a numerical scale for assessingperceived dyspnea (breathing discomfort). It measures the degree ofbreathlessness after completion of the 6 minute walk test (6MWT), wherea BDI of 0 indicates no breathlessness and 10 indicates maximumbreathlessness.

In various aspects, an effective amount of a PH therapy adjusts one ormore hemodynamic parameters indicative of the pulmonary hypertensioncondition towards a more normal level. In one such aspect, mean PAP islowered, for example by at least about 3 mmHg, or at least about 5 mmHg,versus baseline. In another such aspect, PVR is lowered. In yet anothersuch aspect, PCWP or LVEDP is raised.

In various aspects, an effective amount of a PH therapy improvespulmonary function versus baseline. Any measure of pulmonary functioncan be used; illustratively 6MWD is increased or BDI is lowered.

In one such aspect, 6MWD is increased from baseline by at least about 10m, for example at least about 20 m or at least about 30 m. In manyinstances, the method of the present embodiment will be found effectiveto increase 6MWD by as much as 50 m or even more.

In another such aspect, BDI, illustratively as measured following a6MWT, is lowered from baseline by at least about 0.5 index points. Inmany instances, the method of the present embodiment will be foundeffective to lower BDI by as much as 1 full index point or even more.

The SF-36® health survey provides a self-reporting, multi-item scalemeasuring eight health parameters: physical functioning, rolelimitations due to physical health problems, bodily pain, generalhealth, vitality (energy and fatigue), social functioning, rolelimitations due to emotional problems, and mental health (psychologicaldistress and psychological well-being). The survey also provides aphysical component summary and a mental component summary.

In various aspects, an effective amount of a PH therapy can improvequality of life of the subject, illustratively as measured by one ormore of the health parameters recorded in an SF-36® survey. For example,an improvement versus baseline is obtained in at least one of the SF-36physical health related parameters (physical health, role-physical,bodily pain and/or general health) and/or in at least one of the SF-36mental health related parameters (vitality, social functioning,role-emotional and/or mental health). Such an improvement can take theform of an increase of at least 1, for example at least 2 or at least 3points, on the scale for any one or more parameters.

B. Improvement of Prognosis

In another embodiment, the treatment method of the present disclosurecan improve the prognosis for a subject having a pulmonary hypertensioncondition. The treatment of this embodiment can provide (a) a reductionin probability of a clinical worsening event during the treatmentperiod, and/or (b) a reduction from baseline in serum brain natriureticpeptide (BNP) or NT pro-BNP or its N-terminal prohormone, NT-pro-BNPconcentration, wherein, at baseline, time from first diagnosis of thecondition in the subject is not greater than about 2 years.

Time from first diagnosis, in various aspects, can be, for example, notgreater than about 1.5 years, not greater than about 1 year, not greaterthan about 0.75 year or not greater than about 0.5 year. In one aspect,administration of ambrisentan can begin substantially immediately, forexample, within about one month or within about one week, upondiagnosis.

In this embodiment, the treatment period is long enough for the statedeffect to be produced. Typically, the longer the treatment continues thegreater and more lasting will be the benefits. Illustratively, thetreatment period can be at least about one month, for example at leastabout 3 months, at least about 6 months or at least about 1 year. Insome cases, administration can continue for substantially the remainderof the life of the subject.

Clinical worsening event (CWEs) include death, lung transplantation,hospitalization for the pulmonary hypertension condition, atrialseptostomy, initiation of additional pulmonary hypertension therapy oran aggregate thereof. Therefore, the treatments of the presentdisclosure can be effective to provide a reduction of at least about25%, for example at least about 50%, at least about 75% or at leastabout 80%, in probability of death, lung transplantation,hospitalization for pulmonary arterial hypertension, atrial septostomyand/or initiation of additional pulmonary hypertension therapy duringthe treatment period.

Time to clinical worsening of the pulmonary hypertension condition isdefined as the time from initiation of an ambrisentan treatment regimeto the first occurrence of a CWE.

In another particular aspect, the method is effective to provide areduction from baseline of at least about 15%, for example at leastabout 25%, at least about 50% or at least about 75%, in BNP orNT-pro-BNP concentration.

The pulmonary hypertension condition according to this embodiment cancomprise any one or more of the conditions in the WHO, Venice (2003) orDana Point (2009) classifications described above. In one aspect, thecondition comprises PAH (WHO Group 1), for example idiopathic PAH,familial PAH or PAH associated with another disease.

In various aspects of this embodiment, the subject at baseline exhibitsPH (e.g., PAH) of WHO Class I-IV, for example Class I, Class II, ClassIII or Class IV as described above.

In a more particular embodiment, the subject at baseline has a restingPAP of at least about 25 mmHg, for example at least about 30 mmHg, atleast about 35 mmHg or at least about 40 mmHg.

In various aspects of this embodiment, the subject can experience,during or following the treatment period, at least one of:

(a) adjustment of one or more hemodynamic parameters indicative ofimprovement of the cardiopulmonary hypertension condition towards a morenormal level versus baseline;

(b) improvement in cardiopulmonary function; illustratively an increasein exercise capacity or surrogate thereof (e.g., CPET measures such asVO₂ peak, VE/VCO₂, PETCO₂ and the like) or lowering of BDI versusbaseline;

(c) improvement of one or more quality of life parameters versusbaseline; and/or

(d) maintenance of or movement to a lower WHO functional class.

For example, in one aspect the subject can experience improvement incardiopulmonary function versus baseline. Any measure of cardiopulmonaryfunction can be used; illustratively 6MWD is increased or BDI islowered.

In one such aspect, 6MWD is improved from baseline by at least about 10m, for example, at least about 20 m or at least about 30 m. In manyinstances, the method of the present embodiment will be found effectiveto increase 6MWD by as much as 50 m or even more.

In another such aspect, BDI, illustratively as measured following a6MWT, is lowered from baseline by at least about 0.5 point. In manyinstances, the method of the present embodiment will be found effectiveto lower BDI by as much as 1 full index point or even more.

In another aspect, the subject can experience improvement in quality oflife, illustratively as measured by one or more of the health parametersrecorded in an SF-36® survey. For example, an improvement versusbaseline can be obtained in at least one of the SF-36 physical healthrelated parameters (physical health, role-physical, bodily pain and/orgeneral health) and/or in at least one of the SF-35 mental healthrelated parameters (vitality, social functioning, role-emotional and/ormental health). Such an improvement can take the form of an increase ofat least 1, for example at least 2 or at least 3 points, on the scalefor any one or more parameters.

In another aspect, the subject can experience maintenance or improvementin WHO functional class.

C. Prolongation of Life

In yet another embodiment, the treatment methods of the presentdisclosure can prolong the life of a subject having a pulmonaryhypertension condition, from a time of initiation of treatment, by atleast about 30 days. Variants and illustrative modalities of this methodare as set forth above.

D. Extending Time to Clinical Worsening

Still in another embodiment, the present methods can extend time toclinical worsening in a subject having a pulmonary hypertensioncondition, and decrease the probability of a clinical worsening event byat least about 25%. Variants and illustrative modalities of this methodare as set forth above.

E. Other Treatment Objectives

In any of the methods described hereinabove, the subject can be male orfemale. For example, the combined drugs can be administered to a femalesubject according to any of the above methods, including the indicatedvariants and illustrative modalities thereof. Alternatively, ambrisentancan be administered to a male subject, for example a reproductivelyactive male subject, according to any of the above methods, includingthe indicated variants and illustrative modalities thereof.

In another embodiment, the methods provided herein are useful fortreating a pulmonary hypertension condition in a reproductively activemale subject, wherein fertility of the subject is not substantiallycompromised. “Not substantially compromised” in the present contextmeans that spermatogenesis is not substantially reduced by the treatmentand that no hormonal changes are induced that are indicative of orassociated with reduced spermatogenesis. Male fertility can be assesseddirectly, for example, by sperm counts from semen samples, or indirectlyby changes in hormones such as follicle stimulating hormone (FSH),luteinizing hormone (LH), inhibin B and testosterone.

In one embodiment, a method is provided for treating PAH in a subject,wherein the PAH is associated with one or more of (a) a congenital heartdefect, (b) portal hypertension, (c) use of a drug or toxin other thanan anorexigen, (d) thyroid disorder, (e) glycogen storage disease, (f)Gaucher disease, (g) hereditary hemorrhagic telangiectasia, (h)hemoglobinopathy, (i) myeloproliferative disorder, (j) splenectomy, (k)pulmonary veno-occlusive disease and/or (l) pulmonary capillaryhemangiomatosis. Variants and illustrative modalities of this method areas set forth hereinabove.

Further, in another embodiment, a method is provided for treating apulmonary hypertension condition classified in WHO Groups 2-5 in asubject. Variants and illustrative modalities of this method are as setforth hereinabove. In one aspect, the condition comprises left-sidedatrial or ventricular heart disease and/or left-sided valvular heartdisease. In another aspect, the condition is associated with one or moreof chronic obstructive pulmonary disease (COPD), interstitial lungdisease (ILD), sleep-disordered breathing, an alveolar hypoventilationdisorder, chronic exposure to high altitude, a developmentalabnormality, thromboembolic obstruction of proximal and/or distalpulmonary arteries, a non-thrombotic pulmonary embolism, sarcoidosis,histiocytosis X, lymphangiomatosis, and/or compression of pulmonaryvessels.

2.3 Other Uses of the Combinations

Increased or abnormal release of endothelin causes persistentvasoconstriction in the peripheral, renal and cerebral blood vessels,which may lead to illnesses. It has been reported in the literature thatelevated plasma levels of endothelin were found in patients withhypertension, acute myocardial infarct, pulmonary hypertension,Raynaud's syndrome, atherosclerosis and in the airways of asthmatics(see, e.g., U.S. Pat. No. 7,601,730). Accordingly, substances whichspecifically inhibit the binding of endothelin to the receptor oughtalso to antagonize the various abovementioned physiological effects ofendothelin and therefore be valuable drugs, such as ambrisentan andtadalafil. Likewise, the combination of such drugs can also be effectivein treating such diseases and conditions.

Thus, in one aspect, the present disclosure provides a method fortreating or preventing a disease in a patient in need thereof comprisingadministering to the patient therapeutic amounts of a selective type-AERA and a PDE5 inhibitor, wherein the disease is selected from the groupconsisting of hypertension, pulmonary hypertension, myocardialinfarction, angina pectoris, acute kidney failure, renal insufficiency,cerebral vasospasms, cerebral ischemia, subarachnoid hemorrhages,asthma, atherosclerosis, intravascular coagulation, restenosis afterangioplasty, hypertension caused by ischemia or intoxication, kidneyfailure caused by ischemia or intoxication, Raynaud's syndrome andasthmatic airway condition.

Also provided is a method for inhibiting vasoconstriction in a patientin need thereof comprising administering to the patient therapeuticamounts of ambrisentan and tadalafil or pharmaceutically acceptable saltthereof. In one aspect, the vasoconstriction is endothelin-induced.

It is also contemplated that by combining ambrisentan and tadalafil anyundesired side effects may be reduced. For example, administration ofambrisentan to a patient already receiving tadalafil therapy reduces theside effects of tadalafil. The co-action effect of combinedadministration will allow for a reduction in amount of tadalafilnecessary to achieve a therapeutic effect, thereby resulting in areduced incidence of undesirable side effects. As such, in oneembodiment, the disclosure is directed to a method for reducing theundesirable side effects of tadalafil or a salt thereof comprisingadministering a therapeutic amount of ambrisentan or a salt thereof.

As discussed above, by administration of ambrisentan, thetherapeutically effective amount of tadalafil is reduced. As such, thedisclosure, in one embodiment, is directed to a method for reducing thetherapeutically effective dose of tadalafil or a salt thereof comprisingadministering to a patient a therapeutic amount of ambrisentan or a saltthereof.

2.3 Methods

For all of the methods just described, at least one of eitherambrisentan or a salt thereof or tadalafil or a salt thereof isadministered in a less than standard therapeutic dose which becomestherapeutically effective as a consequence of its administration withthe other drug. However, it is also contemplated that tadalafil andambrisentan may also both be administered in a therapeutically effectiveamount. In some embodiments, the tadalafil is administered in aneffective dose and ambrisentan is administered in a standardtherapeutically effective dose. In other embodiment, ambrisentan isadministered in a less than standard therapeutic dose and tadalafil isadministered in a standard therapeutically effective dose. In stillother embodiments, both ambrisentan and tadalafil are administered inless than standard therapeutic doses. The expression “therapeuticamounts of tadalafil and ambrisentan or a salt thereof” is intended toencompass all possible combinations of standard and less than standardtherapeutic doses of ambrisentan and its therapeutically acceptable saltand tadalafil or its therapeutically acceptable salt.

In some embodiments, tadalafil or a salt thereof and ambrisentan or asalt thereof are administered separately or sequentially within a timeperiod effective to provide enhanced efficacy.

Ambrisentan and tadalafil may be given to the patient in either singleor multiple doses by any of the accepted modes of administration ofagents having similar utilities, for example as described in thosepatents and patent applications incorporated by reference, includingbuccal, by intra-arterial injection, intravenously, intraperitoneally,parenterally, intramuscularly, subcutaneously, orally, or via animpregnated or coated device such as a stent, for example, or anartery-inserted cylindrical polymer. In one embodiment, ambrisentan or asalt thereof and tadalafil or a salt thereof are administeredintravenously.

In one embodiment, ambrisentan or a salt thereof and tadalafil or a saltthereof are administered orally. Tadalafil or a salt thereof andambrisentan or a salt thereof may also be administered as a combineddosage unit, such as, for example, in a tablet.

As mentioned above, tadalafil or a salt thereof and ambrisentan or asalt thereof may be administered in a therapeutic amount or an effectiveamount. Therefore, in some embodiments, the amount of ambrisentan or asalt thereof administered is from about 0.5 mg to about 100 mg daily orfrom about 1 mg to about 100 mg daily, or from about 2 mg to about 50 mgdaily, or from about 2 mg to about 20 mg daily. Further, the amount oftadalafil or a salt thereof administered is from about 1 mg to about 500mg daily or from about 5 mg to about 500 mg daily, or from about 5 mg toabout 200 mg daily, or from about 10 mg to about 200 mg daily, or fromabout 10 mg to about 100 mg daily. These aggregate daily doses may beadministered to the patient either once or twice a day.

In one embodiment, whether ambrisentan and tadalafil are administeredtogether or separately, the ratio of the amount of ambrisentan or a saltthereof and the amount of tadalafil or a salt thereof can be in a rangefrom about 1:1 to about 1:10, or alternatively from about 1:1 to about1:8, or alternatively from about 1:2 to about 1:5, or alternatively fromabout 1:2.5 to about 1:3.5 or in a particular aspect, is about 1:3.

Additionally, ambrisentan or a salt thereof is administered as asustained release formulation and/or tadalafil or a salt thereof isadministered as an immediate release or sustained release formulation.This is more thoroughly discussed in the next section.

In one embodiment then, the patient under treatment is already taking amaintenance dose of tadalafil ranging from 20 to 40 mg with a typicaldose once daily. To this dosing regimen is then added ambrisentan atfrom about 5 mg to about 10 mg. By administering such therapeutic dosesof ambrisentan the amount of tadalafil can then be decreased to fromabout 20-40 to about 15-30 mg or about 10-20 mg daily thereby greatlyreducing the incidence of adverse events. Likewise, by administeringsuch therapeutic doses of tadalafil the amount of ambrisentan can thenbe decreased to from about 5-10 to about 3-8 mg or about 2-5 mg dailythereby greatly reducing the incidence of adverse events.

3. Active Ingredients and Compositions 3.1 Pharmaceutical Formulations

As mentioned above, a combination of tadalafil and ambrisentan may beformulated separately. The separate dosage forms containing each activeingredient can be administered sequentially or at similar times (i.e.,either together or one after the other). In another embodiment,tadalafil and ambrisentan is co-formulated into a combined dosage unit.Accordingly, in one embodiment, the disclosure is directed topharmaceutical formulations comprising a therapeutic amount of tadalafilor a salt thereof, a therapeutic amount of ambrisentan or a saltthereof, and a pharmaceutically acceptable carrier.

In another embodiment, the formulation comprises an effective amount ofambrisentan or a salt thereof and/or tadalafil or a salt thereof. Incertain embodiments, the formulations are formulated for eitherintravenous or oral administration. In still other embodiment, the twoactive ingredients are co-formulated into a combined dosage unit. Instill yet other embodiments, the two active ingredients are formulatedseparately for co-therapy administration.

3.2 Co-Formulations

In certain embodiments of the present disclosure, the ambrisentan andtadalafil are co-formulated into a combined dosage unit or unitarydosage form suitable for oral administration. In certain embodiments,the ambrisentan is formulated as a sustained release formulation. Incertain embodiments, the tadalafil is formulated for immediate releaseor sustained release.

In one embodiment, the formulation is in tablet form or capsule form. Inembodiment, the tablet or capsule comprises from about 1 mg to about of500 mg of tadalafil or a pharmaceutically acceptable salt thereof. Inanother embodiment, the tablet or capsule comprises from about 5 mg toabout 500 mg of tadalafil or a pharmaceutically acceptable salt thereof.In yet another embodiment, the tablet or capsule comprises from about 5mg to about 200 mg of tadalafil or a pharmaceutically acceptable saltthereof In still yet another embodiment, the tablet or capsule comprisesfrom about 10 mg to about 200 mg of tadalafil or a pharmaceuticallyacceptable salt thereof. In still yet another embodiment, the tablet orcapsule comprises from about 10 mg to about 100 mg of tadalafil or apharmaceutically acceptable salt thereof

In one embodiment, the tablet or capsule comprises from about 0.5 mg toabout 100 mg of ambrisentan or a pharmaceutically acceptable saltthereof. In another embodiment, the tablet or capsule comprises fromabout 1 mg to about 100 mg of ambrisentan or a pharmaceuticallyacceptable salt thereof. In yet another embodiment, the tablet orcapsule comprises from about 2 mg to about 50 mg of ambrisentan or apharmaceutically acceptable salt thereof. In yet another embodiment, thetablet or capsule comprises from about 2 mg to about 20 mg ofambrisentan or a pharmaceutically acceptable salt thereof.

In one embodiment, the ratio of the amount of ambrisentan or a saltthereof and the amount of tadalafil or a salt thereof, in theformulation, can be from about 1:1 to about 1:10, or alternatively fromabout 1:1 to about 1:8, or alternatively from about 1:2 to about 1:5, oralternatively from about 1:2.5 to about 1:3.5 or in a particular aspect,is about 1:3.

In one such embodiment, the ambrisentan composition is placed in aportion of the tablet which is separate from, but in contact with, theportion of the tablet containing the tadalafil composition. It will beunderstood that the unitary dosage form may comprise simply compressingthe ambrisentan composition and the tadalafil composition into amultilayer tablet or conventionally processed into other conventionalunitary dosage forms such as a capsules. The multilayer tablets andcapsules suitable for use in the present disclosure can be fabricatedusing methods known in the art using standard machinery.

The tablets may comprise two layers, i.e. a first layer which comprisesthe tadalafil and is formulated for immediate release or sustainedrelease, and a second layer which comprises the ambrisentan and isformulated for sustained release. Alternatively, the multilayer tabletmay comprise an inner layer and an outer layer, where the inner layercomprises the sustained release ambrisentan formulation and where theouter layer comprises the immediate release or sustained releasetadalafil layer. In another embodiment, the ambrisentan and tadalafilare co-formulated into a capsule, where the capsule allows for theimmediate release or sustained release of tadalafil and the sustainedrelease of ambrisentan. For example, the capsule may contain granules ofboth tadalafil and ambrisentan, where the granules have been formulatedsuch that the tadalafil is available for immediate release or sustainedrelease and the Ambrisentan is formulated for sustained release.Alternatively, the capsule may contain a liquid immediate release orsustained release formulation of tadalafil and a solid sustained releaseformulation of ambrisentan. However, such embodiments are exemplary andare not intended to limit the formulations of the present disclosure.

A multilayer tablet can be made by compression or molding, optionallywith one or more accessory ingredients. Compressed tablets may beprepared by compressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface active agent ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered active ingredient moistened with aninert liquid diluent. The tablets may optionally be coated or scored.

The tablets may contain one or more agents including sweetening agents,flavoring agents, coloring agents and preserving agents, in order toprovide a palatable preparation. Tablets containing the activeingredients in admixture with non-toxic pharmaceutically acceptableexcipients which are suitable for manufacture of tablets are acceptable.These excipients may be, for example, inert diluents, such as calcium orsodium carbonate, lactose, lactose monohydrate, croscarmellose sodium,povidone, calcium or sodium phosphate; granulating and disintegratingagents, such as maize starch, or alginic acid; binding agents, such ascellulose, microcrystalline cellulose, starch, gelatin or acacia; andlubricating agents, such as magnesium stearate, stearic acid or talc.Tablets may be uncoated or may be coated by known techniques includingmicroencapsulation to delay disintegration and adsorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

3.3 Additional Formulations

Formulations also contemplated by the present disclosure may also be foradministration by injection include aqueous or oil suspensions, oremulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, aswell as elixirs, mannitol, dextrose, or a sterile aqueous solution, andsimilar pharmaceutical vehicles. Aqueous solutions in saline are alsoconventionally used for injection, but less preferred in the context ofthe present disclosure. Ethanol, glycerol, propylene glycol, liquidpolyethylene glycol, and the like (and suitable mixtures thereof),cyclodextrin derivatives, and vegetable oils may also be employed. Theproper fluidity can be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. The prevention ofthe action of microorganisms can be brought about by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. The sameformulations are contemplated for separate administration of ambrisentanand tadalafil.

Sterile injectable solutions are prepared by incorporating the componentin the required amount in the appropriate solvent with various otheringredients as enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

The ideal forms of the apparatus for administration of the novelcombinations for pulmonary hypertension and other methods of thedisclosure consist therefore of (1) either a syringe comprising 2compartments containing the 2 active substances ready for use or (2) akit containing two syringes ready for use.

In making a pharmaceutical composition including ambrisentan andtadalafil, the active ingredients are usually diluted by an excipient orcarrier and/or enclosed within such a carrier that can be in the form ofa capsule, sachet, paper or other container. When the excipient servesas a diluent, in can be a solid, semi-solid, or liquid material (asabove), which acts as a vehicle, carrier or medium for the activeingredient. Thus, the compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosols (as a solid or in a liquid medium),ointments containing, for example, up to 10% by weight of the activecompounds, soft and hard gelatin capsules, sterile injectable solutions,and sterile packaged powders.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents.

The compositions of the disclosure can be formulated so as to providequick, sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.As discussed above, given the reduced bioavailabity of ambrisentan,sustained release formulations are generally preferred. Controlledrelease drug delivery systems for oral administration include osmoticpump systems and dissolutional systems containing polymer-coatedreservoirs or drug-polymer matrix formulations. Examples of controlledrelease systems are given in U.S. Pat. Nos. 3,845,770; 4,326,525;4,902,514; and 5,616,345.

The compositions are preferably formulated in a unit dosage form. Theterm “unit dosage forms” or “combined dosage unit” refers to physicallydiscrete units suitable as unitary dosages for human subjects and othermammals, each unit containing a predetermined quantity of the activematerials calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient (e.g., a tablet,capsule, ampoule). The active agents of the disclosure are effectiveover a wide dosage range and are generally administered in apharmaceutically effective amount. It will be understood, however, thatthe amount of each active agent actually administered will be determinedby a physician, in the light of the relevant circumstances, includingthe condition to be treated, the chosen route of administration, theactual compounds administered and their relative activity, the age,weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredients are mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present disclosure. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredients are dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules.

The tablets or pills of the present disclosure may be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action, or to protect from the acid conditions of the stomach.For example, the tablet or pill can comprise an inner dosage and anouter dosage element, the latter being in the form of an envelope overthe former. Ambrisentan and the co-administered agent(s) can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permit the inner element to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol, and cellulose acetate.

Additional embodiments of the disclosure include kits comprising atherapeutic amount of ambrisentan or a salt thereof and a therapeuticamount of tadalafil or a salt thereof.

The following examples are included to demonstrate preferred embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the disclosure, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe disclosure.

EXAMPLES

Tadalafil as used in this disclosure is commercially available and canbe prepared by conventional methods such as in the manner disclosed inU.S. Pat. No. 5,859,006. Ambrisentan is also commercially available ormay be prepared by conventional methods such as in the manner disclosedin U.S. Pat. No. 5,703,017. The entire disclosure of both patents ishereby incorporated by reference. Additionally, the abbreviations usedthroughout have the following meanings:

-   -   μM=micromolar    -   μm=micrometer    -   cm=centimeter    -   kg=kilogram    -   mA=milliamp    -   min=minute    -   mm=millimeter    -   mM=millimolar    -   mpk=milligram per kilogram (body weight)    -   ms=millisecond    -   nM=nanomolar    -   AMB=ambrisentan    -   TAD=tadalafil    -   BOS=bosentan    -   MAC=macitentan    -   ET-1=endothelin-1

Example 1

Ambrisentan and Tadalafil Relax Endothelin-Induced Contraction of RatPulmonary Arteries and Aortas

This example examines the pharmacological effects of the combination ofambrisentan and tadalafil in comparison with either of them alone, withrespect to their capability to relax isolated rat pulmonary artery andthoracic aorta preparations.

The selective type-A endothelin receptor antagonist, ambrisentan(Letairis®), and the phosphodiesterase type 5 inhibitor, tadalafil(Adcirca®), are currently used to treat pulmonary arterial hypertension.Isolated rat intact intrapulmonary arterial rings contracted with 8 nMendothelin-1 (ET-1) were relaxed by 10 nM ambrisentan (from GileadSciences, Inc.) and 30 nM tadalafil (from Sequoia Research Products Ltd,Pangbourne, UK) by 30±13% (mean±SEM, n=3) and 12±5% (n=3), respectively,whereas both drugs in combination relaxed the intact intrapulmonaryarterial rings by 77±5% (FIG. 1, n=3, P<0.01 vs. mono-administration ofambrisentan or tadalafil).

Similarly, mono-administration of 10 nM ambrisentan and 30 nM tadalafilrelaxed isolated rat intact thoracic aortic rings in the presence of 8nM ET-1 by 32±3% (n=3) and 16±4% (n=3), respectively. The combination ofboth 10 nM ambrisentan and 30 nM tadalafil relaxed the ET-1-inducedcontraction of the intact thoracic aortic rings by 81±7% (FIG. 1, n=3;P<0.01 vs. mono-administration of ambrisentan or tadalafil). The IC₅₀value for tadalafil to relax ET-1 constricted aortic rings was reducedfrom 79 nM to 16 nM in the presence of 10 nM ambrisentan.

In endothelium-denuded aortic rings, tadalafil failed to inhibitcontraction induced by ET-1 whereas 10 nM ambrisentan reducedcontraction by 26±7% (n=4); the drug combination was not more effectivethan mono-administration of ambrisentan.

These data suggest that the combination of ambrisentan and tadalafilinhibit endothelin-induced vasoconstriction and endothelium is requiredto produce their enhanced effect on vasorelaxation.

Example 2 Selective Type-A ERA and PDE5 Inhibitor Shows Co-Action inRelaxing Endothelin-Induced Contraction of Pulmonary Arteries whileNon-Selective ERA and PDE5 Inhibitor Lack such Co-Action

This Example confirms the beneficial co-action of ambrisentan andtadalafil as observed in Example 1 and further investigates themechanism underlying such co-action.

Ambrisentan (Letairis®) is a selective type-A endothelin receptorantagonist approved for treatment of PAH. Bosentan (Tracleer®) is anon-selective (types A&B) endothelin receptor antagonist for PAH.Macitentan (second generation of Bosentan) is a non-selective endothelinreceptor antagonist in phase III for PAH. Tadalafil (Adcirca® andCialis®) is a PDE5 inhibitor for PAH and erectile dysfunction (ED).

Method: Ex-Vivo Vascular Function Assay

Intrapulmonary arteries (200-500 μm) and aortas were isolated fromSprague Dawley rats (300-320 g) and cut into 1-2 mm rings. Rings weremounted in a myograph and constricted with a submaximal concentration (8nM) of ET-1. Ambrisentan, Bosentan, Macitentan and Tadalafil alone andin combination were evaluated in ET-constricted rings.

Results:

FIG. 2 shows the effects of Tadalafil with Ambrisentan, Bosentan orMacitentan to attenuate ET-1-induced contraction of rat pulmonary arteryrings.

Isolated rat intact intrapulmonary arterial rings contracted with 8 nMendothelin-1 (ET-1) were relaxed by 10 nM ambrisentan and 30 nMtadalafil by 29±1% (mean±SEM, n=4) and 22±2% (n=4), respectively,whereas both drugs in combination relaxed the intact intrapulmonaryarterial rings by 85±3% (FIG. 2, n=4, P<0.01 vs. mono-administration ofambrisentan or tadalafil).

In contrast, the combination of 30 nM tadalafil with 100 nM bosentan or30 nM macitentan, two nonselective type-A & B endothelin receptorantagonists, relaxed the ET-1-depdendent contraction of the intactintrapulmonary arterial rings by 50±4% (FIG. 2, n=4, P<0.05 vs.mono-administrations of bosentan and tadalafil or combination ofambrisentan with tadalafil) or 48±8% (FIG. 2, n=7, P<0.05 vs.mono-administrations of macitentan and tadalafil or combination ofambrisentan with tadalafil). Mono-administrations of 100 nM bosentan and30 nM macitentan produced a vasorelaxant effect similar to 10 nMambrisentan and relaxed the isolated intact intrapulmonary arterialrings contracted with 8 nM ET-1 by 25±1% (n=4) and 31±2% (n=4),respectively.

FIG. 3 shows the effects of TAD with AMB or BOS to attenuateET-1-induced contraction of rat aortic rings. Again, the effect of thecombination of AMB and TAD was almost double of the sum ofmono-administration of each drug. In contrast, such enhancedeffectiveness was not observed for the combination of TAD and BOS.

In FIG. 4, the contraction graphs show the effects of TAD in combinationwith AMB or BOS on ET-1-induced contraction of rat aortic rings (up: notreatment; middle: AMB+TAD; down: BOS+TAD). Apparently, the combinationof AMB and TAD is much more effective than the combination of BOS andTAD.

In endothelium-denuded pulmonary arterial rings, 30 nM tadalafil failedto inhibit contraction induced by ET-1 whereas 10 nM ambrisentan reducedcontraction by 23±4% (n=4); the drug combination was not more effectivethan mono-administration of ambrisentan (FIG. 5). FIG. 5 therefore showsthat endothelium is involved in additive effect of ET Receptorantagonists with TAD.

In the presence of 1 μM BQ-788, a selective type-B endothelin receptorantagonist, the combination effect of 10 nM ambrisentan with 30 nMtadalafil was significantly reduced from 85±3% (n=4) to 56±7% (FIG. 6,n=4, P<0.05 vs. combination of ambrisentan with tadalafil) which isclose to the additive effect of 30 nM tadalafil with 100 nM bosentan or30 nM macitentan. BQ-788 did not have an effect on the relaxation bycombination of tadalafil with bosentan (FIG. 6).

These data suggest that the co-action of ambrisentan and tadalafilinhibiting endothelin-induced vasoconstriction and the type-B endothelinreceptor in endothelium contributes to their enhanced effect onvasorelaxation. The mechanism is explained and illustrated in FIG. 7-9.FIG. 7 illustrates the ET-1 and PDE5 signaling in endothelial cells andsmooth muscle cells. A selective type-A ERA such as AMB and a PDE5inhibitor such as TAD target vasorelaxation from two different pathwaysleading to enhanced effectiveness (FIG. 8), whereas a non-selective ERAor a selective type-B ERA and a PDE5 inhibitor would not have suchbenefits because they target the same pathway (FIG. 9).

Based on this mechanism, therefore, the co-action observed in thepresent example based on the combination of ambrisentan and tadalafilcan be extrapolated to other selective type-A ERA's and PDE5 inhibitors.

Example 3 Selective Type-A ERA and PDE5 Inhibitor AttenuatedHypoxia-Induced Pulmonary Arterial Hypertension (PAH)

This Example demonstrates the co-action of ambrisentan and tadalafil ina pulmonary arterial hypertension (PAH) animal model.

Method: Pulmonary Arterial Hypertension (PAH) Animal Model

Male SD Rats (225-250 g) were housed in chambers under normoxic (sealevel) or hypoxic (10% oxygen) conditions for 3 weeks.

Rats were dosed with vehicle (0.5% hydroxypropyl methylcellulose (HPMC),0.2% Tween 80, and 0.9% benzyl alcohol in water), AMB or TAD (quaquedie) beginning the day they were placed in chambers. Plasma wascollected when animals were terminated. Table 1 lists the animals usedin this study, along with the treatments they received.

TABLE 1 Group N Manipulation Treatment Duration 1 8 Normoxia Vehicle 3wks 2 12 Hypoxia Vehicle 3 wks 3 12 Hypoxia 10 mpk TAD 3 wks 4 12Hypoxia 1 mpk AMB 3 wks 5 12 Hypoxia 10 mpk AMB 3 wks 6 12 Hypoxia 1 mpkAMB + 10 mpk TAD 3 wks 7 12 Hypoxia 10 mpk AMB + 10 mpk TAD 3 wks

For AMB, 1 mg per kg body weight (mpk) resulted in about 17 nM free orunbounded plasma concentration when administered separately, or about 13nM free or unbounded plasma concentration when administered along withTAD. The free or unbounded plasma concentration for 10 mpk AMB dosingwas about 104 nM in both cases. The free or unbounded plasmaconcentrations of TAD was about 24 nM when administered separately, orabout 26 nM when administered with AMB, whether 1 mpk or 10 mpk.

Results:

Compared to animals treated with the vehicle, treatment withmono-administration of TAD or AMB inhibited hypoxia-induced meanpulmonary arterial pressure (mPAP) (FIG. 10). TAD, 1 mpk AMB and 10 mpkAMB showed 23.2±1.3%, 28.4±1.1% and 42.1±2.4% inhibition of PAP(mPAP±SEM), respectively.

When administered together, TAD and 1 mpk AMB led to a 60.8±3.7% PAPinhibition, which was statistically significantly greater than theadditive effect of mono-administration of each agent (51.6±1.8%,p<0.05). Likewise, the co-administration of TAD and 10 mpk AMB achieveda 71.7±2.3% PAP inhibition, which was also statistically significantlygreater than the additive effect of mono-administration of each agent(65.3±1.9%, p<0.05).

Therefore, this example demonstrates, in vivo, the co-action ofambrisentan and tadalafil in inhibiting PAH.

What is claimed is: 1-32. (canceled)
 33. A composition comprising ambrisentan and tadalafil, wherein the weight ratio of the ambrisentan to the tadalafil is about 1:2 or about 1:3.
 34. The composition of claim 1, wherein the weight ratio of the ambrisentan to the tadalafil is about 1:2.
 35. The composition of claim 1, wherein the ambrisentan to the tadalafil are co-formulated.
 36. The composition of claim 1, which is formulated for parenteral administration.
 37. The composition of claim 1, which is formulated for oral administration.
 38. The composition of claim 37, which is in a tablet form or capsule form.
 39. The composition of claim 1, wherein the amount of ambrisentan is from about 1 mg to about 100 mg.
 40. The composition of claim 1, wherein the amount of ambrisentan is from about 2 mg to about 20 mg.
 41. The composition of claim 1, wherein the amount of tadalafil is from about 5 mg to about 500 mg.
 42. The composition of claim 1, wherein the amount of tadalafil is from about 10 mg to about 100 mg.
 43. The composition of claim 1, further comprising a drug selected from the group consisting of prostanoids, PDE5 inhibitors other than tadalafil, endothelin receptor antagonists other than ambrisentan, calcium channel blockers, diuretics, anticoagulants, oxygen and combinations thereof.
 44. A tablet or capsule comprising ambrisentan and tadalafil, wherein the weight ratio of the ambrisentan to the tadalafil is about 1:2.
 45. The tablet or capsule of claim 44, comprising about 5 mg ambrisentan and about 10 mg tadalafil.
 46. The tablet or capsule of claim 44, comprising about 10 mg ambrisentan and about 20 mg tadalafil.
 47. The tablet or capsule of claim 44, wherein the ambrisentan and the tadalafil are co-crystallized.
 48. The tablet or capsule of claim 44, further comprising a drug selected from the group consisting of prostanoids, PDE5 inhibitors other than tadalafil, endothelin receptor antagonists other than ambrisentan, calcium channel blockers, diuretics, anticoagulants, oxygen and combinations thereof. 